Bone morphogenetic protein 5(BMP-5) compositions

ABSTRACT

Purified BMP-5 proteins and processes for producing them are disclosed. The proteins may be used in the treatment of bone and/or cartilage defects and in wound healing and related tissue repair.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 07/995,565 filedDec. 22, 1992 now abandoned which is a continuation of U.S. Ser. No.07/588,227 filed Sep. 26, 1990 abandoned which is continuation-in-partof U.S. Ser. No. 437,409 filed Nov. 15, 1989 now abandoned; Ser. No.370,547 filed Jun. 23, 1989 now U.S. Pat. No. 5,106,748; Ser. No.347,559 filed May 4, 1989, now abandoned; and Ser. No. 329,610 filed 28Mar. 1989, now abandoned which is a continuation-in-part of U.S. Ser.Nos. 179,100, now U.S. Pat. No. 5,013,649; 179,101, abandoned; and179,197, abandoned, each filed 8 Apr. 1988 which arecontinuations-in-part of U.S. Ser. Nos. 028,285 filed Mar. 20, 1987 nowabandoned and 031,346 filed Mar. 26, 1987 now U.S. Pat. No. 4,877,864which are continuations-in-part of U.S. Ser. Nos. 943,332 filed Dec. 17,1986 now abandoned and 880,776 filed Jul. 1, 1986 now abandoned.

The present invention relates to a family of purified proteins, termedBMP-5 proteins (wherein BMP is bone morphogenic protein), which exhibitthe ability to induce cartilage and/or bone formation and processes forobtaining them. These proteins may be used to induce bone and/orcartilage formation and in wound healing and tissue repair.

The invention provides human BMP-5 proteins, substantially free fromother proteins with which they are co-produced, comprising the aminoacid sequence set forth in Table III from amino acid #323 (Asn, Gln,Asn) to amino acid #454 (ending with Gly, Cys, His). This amino acidsequence #323 to #454 is encoded by the DNA sequence of Table III fromnucleotide #1665 to nucleotide #2060. The mature BMP-5 dimer may befurther characterized by an apparent molecular weight of approximately28,000-38,000 daltons as determined by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE). Under reducing conditionsin SDS-PAGE the mature subunit electrophoreses with a molecular weightof approximately 18,000-22,000 daltons. These proteins are capable ofstimulating, promoting, or otherwise inducing cartilage and/or boneformation.

The invention further provides bovine BMP-5 proteins comprising theamino acid sequence set forth in Table I from #9 to amino acid #140. Theamino acid sequence from #9 to #140 is encoded by the DNA sequence fromnucleotide #32 to #427 of Table I. These proteins may be furthercharacterized by an apparent molecular weight of 28,000-30,000 daltonsas determined by sodium dodecyl sulfate polyacrylamide gelelectrophoresis (SDS-PAGE). Under reducing conditions in SDS-PAGE theprotein electrophoreses with a molecular weight of approximately14,000-20,000 daltons. It is contemplated that these proteins arecapable of inducing cartilage and/or bone formation.

Human BMP-5 proteins of the invention may be produced by culturing acell transformed with a DNA sequence containing the nucleotide sequencethe same or substantially the same as the nucleotide sequence shown inTable III comprising nucleotide #699 to nucleotide #2060. BMP-5 proteinscomprising the amino acid sequence the same or substantailly the same asshown in Table III from amino acid #323 to #454 are recovered isolatedand purified from the culture media.

Bovine proteins of the invention may be produced by culturing a celltransformed with a DNA sequence containing the nucleotide sequence thesame or substantially the same as that shown in Table I comprisingnucleotide #8 through nucleotide #427 and recovering and purifying fromthe culture medium a protein containing the amino acid sequence or aportion thereof as shown in Table I comprising amino acid #9 to aminoacid #140.

The invention further provides a method wherein the proteins describedabove are utilized for obtaining related human protein/s or othermammalian cartilage and/or bone growth protein/s. Such methods are knownto those skilled in the art of genetic engineering. One method forobtaining such proteins involves utilizing the human BMP-5 codingsequence or portions thereof from nucleotide #699-#2060 as a probe forscreening human genomic and/or cDNA libraries to isolate the humangenomic and/or cDNA sequence. Additional methods known in the art mayemploy the bovine and human BMP-5 proteins of the invention to obtainother mammalian BMP-5 cartilage and/or bone formation proteins.

Having identified the nucleotide sequences the proteins are produced byculturing a cell transformed with the DNA identified in the methoddescribed above which DNA hybridizes under stringent conditions to thebovine BMP-5 nucleotide sequence substantially as shown in Table I orthe human BMP-5 nucleotide sequence substantially as shown in Table IIIand which encodes a protein exhibiting cartilage and/or bone formationactivity. The expressed proteins are recovered and purified from theculture media. The purified BMP-5 proteins are substantially free fromother proteinaceous materials with which they are co-produced, as wellas from other contaminants.

The BMP-5 proteins of the invention are characterized by the ability topromote, stimulate or otherwise induce the formation of cartilage and/orbone. It is further contemplated that the ability of these proteins toinduce the formation of cartilage and/or bone may be exhibited by theability to demonstrate cartilage and/or bone formation activity in therat bone formation assay described below. It is further contemplatedthat the proteins of the invention may demonstrate activity in this ratbone formation assay at a concentration of 10 μg-500 μg/gram of bone.More particularly, it is contemplated these proteins may becharacterized by the ability of 1 μg of the protein to score at least +2in the rat bone formation assay described below using either theoriginal or modified scoring method.

Another aspect of the invention provides pharmaceutical compositionscontaining a therapeutically effective amount of a BMP-5 protein of theinvention in a pharmaceutically acceptable vehicle or carrier. Thecompositions of the invention may be used to induce bone and/orcartilage formation. These compositions may also be used for woundhealing and tissue repair. Further compositions of the invention mayinclude in addition to a BMP-5 protein of the present invention at leastone other therapeutically useful agent such as the proteins designatedBMP-1, BMP-2A and -2B, BMP-3, BMP-6, and BMP-7 disclosed respectively inco-owned U.S. patent applications Ser. No. 179,101, abandoned in favorof Ser. No. 561,496 now U.S. Pat. No. 5,108,922, Ser. No. 179,100 nowU.S. Pat. No. 5,013,649, and Ser. No. 179,197 abandoned in favor of Ser.No. 692,827 now U.S. Pat. No. 5,116,738, Ser. No. 370,544, and Ser. No.370,549 abandoned in favor of Ser. No. 438,919 now U.S. Pat. No.5,141,905. These proteins may act in concert with or perhapssynergistically with one another. Other therapeutically useful agentsmay include growth factors such as epidermal growth factor (EGF),fibroblast growth factor (FGF), transforming growth factors (TGF-α andTGF-β), and platelet derived growth factor (PDGF).

The compositions of the invention may also include an appropriatematrix, for instance, for delivery and/or support of the compositionand/or providing a surface for bone and/or cartilage formation. Thematrix may provide slow release of the BMP-5 proteins and/or theappropriate environment for presentation of the BMP-5 proteins of theinvention.

The compositions may be employed in methods for treating a number ofbone and/or cartilage defects, and periodontal disease. They may also beemployed in methods for treating various types of wounds and in tissuerepair. These methods, according to the invention, entail administeringto a patient needing such bone and/or cartilage formation, wound healingor tissue repair, a therapeutically effective amount of a protein of theinvention. These methods may also entail the administration of a proteinof the invention in conjunction with at least one of the "BMP" proteinsdisclosed in the co-owned applications described above. In addition,these methods may also include the administration of a protein of theinvention with other growth factors including EGF, FGF, TGF-α, TGF-β,and PDGF.

Still a further aspect of the invention are DNA sequences coding forexpression of a protein of the invention. Such sequences include thesequence of nucleotides in a 5' to 3' direction illustrated in Table Ior Table III or DNA sequences which hybridize under stringent conditionswith the DNA sequence of Table I or Table III and encode a proteindemonstrating ability to induce cartilage and/or bone formation as inthe rat bone formation assay described below. It is contemplated thatthese proteins may demonstrate activity in this assay at a concentrationof 10 μg-500 μg/gram of bone. More particularly, it is contemplated thatthese proteins demonstrate the ability of 1 μg of the protein to scoreat least +2 in the rat bone formation assay using either the original ormodified scoring method. Finally, allelic or other variations of thesequences of Table I and III whether such nucleotide changes result inchanges in the peptide sequence or not, are also included in the presentinvention.

A further aspect of the invention provides a vector containing a DNAsequence as described above in operative association with an expressioncontrol sequence therefor. These vectors may be employed in a novelprocess for producing a protein of the invention in which a cell linetransformed with a DNA sequence directing expression of a protein of theinvention in operative association with an expression control sequencetherefor, is cultured in a suitable culture medium and a protein of theinvention is recovered and purified therefrom. This claimed process mayemploy a number of known cells, both prokaryotic and eukaryotic, as hostcells for expression of the polypeptide. The recovered BMP protiens arepurified by isolating them from other proteinaceous materials with whichthey are co-produced as well as from other contaminants.

Other aspects and advantages of the present invention will be apparentupon consideration of the following detailed description and preferredembodiments thereof.

DETAILED DESCRIPTION OF THE INVENTION

Purified human BMP-5 proteins are produced by culturung a host celltransformed with the DNA sequence of Table III. The expressed BMP-5proteins are isolated and purified from the culture media. The purifiedhuman BMP-5 proteins are characterized by comprising an amino acidsequence as shown in Table III from amino acid #323 to #454. Thesepurified BMP-5 human cartilage/bone proteins of the present inventionmay be produced by culturing a host cell transformed with a DNA sequencecomprising the DNA sequence as shown in Table III from nucleotide #699to nucleotide #2060 or substantially homologous sequences operativelylinked to a heterologous regulatory control sequence and recovering andpurifying from the culture medium a protein comprising the amino acidsequence as shown in Table III from amino acid #323 to amino acid #454or a substantially homologous sequence.

In further embodiments the DNA sequence comprises the nucleotidesencoding amino acids #323-#454 of Table III. BMP-5 proteins maytherefore be produced by culturing a host cell transformed with a DNAsequence comprising the DNA sequence as shown in Table III fromnucleotide #1665 to nucleotide #2060 or substantially homologoussequences operatively linked to a heterologous regulatory controlsequence and recovering and purifying from the culture medium a proteincomprising the amino acid sequence as shown in Table III from amino acid#323 to amino acid #454 or a substantially homologous sequence. Thepurified human BMP-5 proteins are substantially free from otherproteinaceous materials with which they are co-produced, as well as fromother contaminants.

Purified BMP-5 bovine cartilage/bone proteins of the present inventionare produced by culturing a host cell transformed with a DNA sequencecomprising the DNA sequence as shown in Table I from nucleotide #8 tonucleotide #578 or substantially homologous sequences and recovering andpurifying from the culture medium a protein comprising the amino acidsequence as shown in Table I from amino acid #9 to amino acid #140 or asubstantially homologous sequence. The purified BMP-5 bovine proteins ofthe invention are substantially free from other proteinaceous materialswith which they are co-produced, as well as from other contaminants.

BMP-5 proteins are further characterized by the ability to demonstratecartilage and/or bone formation activity. This activity may bedemonstrated, for example, in the rat bone formation assay as describedin Example III. It is further contemplated that these proteinsdemonstrate activity in the assay at a concentration of 10 μg-500μg/gram of bone formed. The proteins may be further characterized by theability of 1 μg to score at least +2 in this assay using either theoriginal or modified scoring method described below.

The mature BMP-5 dimer may be further characterized by an apparentmolecular weight of approximately 28,000-38,000 daltons as determined bysodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).Under reducing conditions in SDS-PAGE the mature sub-unitelectrophoreses with a molecular weight of approximately 18,000-22,000daltons.

The proteins provided herein also include factors encoded by thesequences similar to those of Table I and Table III but into whichmodifications are naturally provided (e.g. allelic variations in thenucleotide sequence which may result in amino acid changes in thepolypeptide) or deliberately engineered. Similarly, syntheticpolypeptides which wholly or partially duplicate continuous sequences ofthe amino acid residues of Table I or Table III are encompassed by theinvention. These sequences, by virtue of sharing primary, secondary, ortertiary structural and conformational characteristics with othercartilage/bone proteins of the invention may possess bone and/orcartilage growth factor biological properties in common therewith. Thus,they may be employed as biologically active substitutes fornaturally-occurring proteins in therapeutic processes.

Other specific mutations of the sequences of the proteins of theinvention described herein may involve modifications of a glycosylationsite. These modification may involve O-linked or N-linked glycosylationsites. For instance, the absence of glycosylation or only partialglycosylation at asparagine-linked glycosylation sites results fromamino acid substitution or deletion at any asparagine-linkedglycosylation recognition site present in the sequences of the proteinsof the invention, for example, as shown in Table I or Table III. Theasparagine-linked glycosylation recognition sites comprise tripeptidesequences which are specifically recognized by appropriate cellularglycosylation enzymes. These tripeptide sequences are eitherasparagine-X-threonine or asparagine-X-serine, where X is usually anyamino acid. A variety of amino acid substitutions or deletions at one orboth of the first or third amino acid positions of a glycosylationrecognition site (and/or amino acid deletion at the second position)results in non-glycosylation at the modified tripeptide sequence.Expression of such altered nucleotide sequences produces variants whichare not glycosylated at that site.

The present invention also encompasses the novel DNA sequences, free ofassociation with DNA sequences encoding other proteinaceous materials,and coding on expression for the proteins of the invention. These DNAsequences include those depicted in Tables I and III in a 5' to 3'direction. Further included are those sequences which hybridize understringent hybridization conditions [see, T. Maniatis et al, MolecularCloning (A Laboratory Manual), Cold Spring Harbor Laboratory (1982),pages 387 to 389] to the DNA sequence of Table I or Table III anddemonstrate cartilage and/or bone formation activity in the rat boneformation assay. An example of one such stringent hybridizationcondition is hybridization at 4×SSC at 65° C., followed by a washing in0.1×SCC at 65° C. for an hour. Alternatively, an exemplary stringenthybridization condition is in 50% formamide, 4×SCC at 42° C.

Similarly, DNA sequences which encode proteins similar to the proteinencoded by the sequence of Table I or Table III, but which differ incodon sequence due to the degeneracies of the genetic code or allelicvariations (naturally-occurring base changes in the species populationwhich may or may not result in an amino acid change) also encode theproteins of the invention described herein. Variations in the DNAsequences of Table I and Table III which are caused by point mutationsor by induced modifications (including insertion, deletion, andsubstitution) to enhance the activity, half-life or production of thepolypeptides encoded thereby are also encompassed in the invention.

In a further aspect, the invention provides a method for obtainingrelated human proteins or other mammalian BMP-5 proteins. One method forobtaining such proteins entails, for instance, utilizing the human BMP-5coding sequence disclosed herein to probe a human genomic library usingstandard techniques for the human gene or fragments thereof. Sequencesthus identified may also be used as probes to identify a human cell lineor tissue which synthesizes the analogous cartilage/bone protein. A cDNAlibrary is synthesized and screened with probes derived from the humanor bovine coding sequences. The human sequence thus identified istransformed into a host cell, the host cell is cultured and the proteinrecovered, isolated and purified from the culture medium. The purifiedprotein is predicted to exhibit cartilage and/or bone formation activityin the rat bone formation assay of Example III.

Another aspect of the present invention provides a novel method forproducing the proteins of the invention. The method of the presentinvention involves culturing a suitable cell line, which has beentransformed with a DNA sequence coding for expression of a protein ofthe invention, under the control of known regulatory sequences.Regulatory sequences include promoter fragments, terminator fragmentsand other suitable sequences which direct the expression of the proteinin an appropriate host cell. Methods for culturing suitable cell linesare within the skill of the art. The transformed cells are cultured andthe BMP-5 proteins expressed thereby are recovered and purified from theculture medium using purification techniques known to those skilled inthe art. The purified BMP-5 proteins are substantially free from otherproteinaceous materials with which they are co-produced, as well asother contaminants. Purified BMP-5 proteins are substantially free frommaterials with which the proteins of the invention exist in nature.

Suitable cells or cell lines may be mammalian cells, such as Chinesehamster ovary cells (CHO). The selection of suitable mammalian hostcells and methods for transformation, culture, amplification, screeningand product production and purification are known in the art. See, e.g.,Gething and Sambrook, Nature, 293:620-625 (1981), or alternatively,Kaufman et al, Mol. Cell. Biol., 5(7):1750-1759 (1985) or Howley et al,U.S. Pat. No. 4,419,446. Other suitable mammalian cell lines are themonkey COS-1 cell line and the CV-1 cell line.

Bacterial cells may also be suitable hosts. For example, the variousstrains of E. coli (e.g., HB101, MC1061) are well-known as host cells inthe field of biotechnology. Various strains of B. subtilis, Pseudomonas,other bacilli and the like may also be employed in this method.

Many strains of yeast cells known to those skilled in the art may alsobe available as host cells for expression of the polypeptides of thepresent invention. Additionally, where desired, insect cells may beutilized as host cells in the method of the present invention. See, e.g.Miller et al, Genetic Engineering, 8:277-298 (Plenum Press 1986) andreferences cited therein.

Another aspect of the present invention provides vectors for use in themethod of expression of the proteins of the invention. The vectorscontain the novel DNA sequences described above which code for the novelcartilage/bone proteins of the invention. Additionally the vectors alsocontain appropriate expression control sequences permitting expressionof the protein sequences. Alternatively, vectors incorporating truncatedor modified sequences as described above are also embodiments of thepresent invention and useful in the production of the proteins of theinvention. The vectors may be employed in the method of transformingcell lines and contain selected regulatory sequences in operativeassociation with the DNA coding sequences of the invention which arecapable of directing the replication and expression thereof in selectedhost cells. Useful regulatory sequences for such vectors are known tothose skilled in the art and may be selected depending upon the selectedhost cells. Such selection is routine and does not form part of thepresent invention. Host cells transformed with such vectors and progenythereof for use in producing cartilage/bone proteins are also providedby the invention.

One skilled in the art can construct mammalian expression vectors byemploying the DNA sequences of the invention and known vectors, such aspCD [Okayama et al., Mol. Cell Biol., 2:161-170 (1982)] and pJL3, pJL4[Gough et al., EMBO J., 4:645-653 (1985)]. Similarly, one skilled in theart could manipulate the sequences of the invention by eliminating orreplacing the mammalian regulatory sequences flanking the codingsequence with bacterial sequences to create bacterial vectors forintracellular or extracellular expression by bacterial cells. Forexample, the coding sequences could be further manipulated (e.g. ligatedto other known linkers or modified by deleting non-coding sequencesthere-from or altering nucleotides therein by other known techniques).The modified coding sequence could then be inserted into a knownbacterial vector using procedures such as described in T. Taniguchi etal., Proc. Natl Acad. Sci. USA, 77:5230-5233 (1980). This exemplarybacterial vector could then be transformed into bacterial host cells anda protein of the invention expressed thereby. For a strategy forproducing extracellular expression of a cartilage and/or bone protein ofthe invention in bacterial cells., see, e.g. European patent applicationEPA 177,343.

Similar manipulations can be performed for the construction of an insectvector [See, e.g. procedures described in published European patentapplication 155,476] for expression in insect cells. A yeast vectorcould also be constructed employing yeast regulatory sequences forintracellular or extracellular expression of the factors of the presentinvention by yeast cells. [See, e.g., procedures described in publishedPCT application WO86/00639 and European patent application EPA 123,289].

A method for producing high levels of a protein of the invention frommammalian cells involves the construction of cells containing multiplecopies of the heterologous gene encoding proteins of the invention. Theheterologous gene may be linked to an amplifiable marker, e.g. thedihydrofolate reductase (DHFR) gene for which cells containing increasedgene copies can be selected for propagation in increasing concentrationsof methotrexate (MTX) according to the procedures of Kaufman and Sharp,J. Mol. Biol., 159:601-629 (1982). This approach can be employed with anumber of different cell types.

For instance, a plasmid containing a DNA sequence for a protein of theinvention in operative association with other plasmid sequences enablingexpression thereof and the DHFR expression plasmid pAdA26SV(A)3 [Kaufmanand Sharp, Mol. Cell. Biol., 2:1304 (1982)] may be co-introduced intoDHFR-deficient CHO cells, DUKX-BII, by calcium phosphate coprecipitationand transfection, electroperation or protoplast fusion. DHFR expressingtransformants are selected for growth in alpha media with dialyzed fetalcalf serum, and subsequently selected for amplification by growth inincreasing concentrations of MTX (sequential steps in 0.02, 0.2, 1.0 and5 uM MTX) as described in Kaufman et al., Mol Cell Biol., 5:1750 (1983).Protein expression should increase with increasing levels of MTXresistance. Transformants are cloned, and the proteins of the inventionare recovered, isolated, and purified from the culture medium.Characterization of expressed proteins may be carried out using stnadardtechniques. For instance, characterization may include pulse labelingwith [35^(S) ] methionine or cysteine, or polyacrylamide gelelectrphoresis. Biologically active protein expression is monitored bythe Rosen-modified Sampath - Reddi rat bone formation assay describedabove in Example III. Similar procedures can be followed to produceother related proteins.

A protein of the present invention, which induces cartilage and/or boneformation in circumstances where bone and/or cartilage is not normallyformed, has application in the healing of bone fractures and cartilagedefects in humans and other animals. Such a preparation employing aprotein of the invention may have prophylactic use in closed as well asopen fracture reduction and also in the improved fixation of artificialjoints. De novo bone formation induced by an osteogenic agentcontributes to the repair of congenital, trauma induced, or oncologicresection induced craniofacial defects, and also is useful in cosmeticplastic surgery. A protein of the invention may be used in the treatmentof periodontal disease, and in other tooth repair processes. Such agentsmay provide an environment to attract bone-forming cells, stimulategrowth of bone-forming cells or induce differentiation of progenitors ofbone-forming cells. A variety of osteogenic, cartilage-inducing and boneinducing factors have been described. See, e.g. European patentapplications 148,155 and 169,016 for discussions thereof.

The proteins of the invention may also be used in wound healing andrelated tissue repair. The types of wounds include, but are not limitedto burns, incisions and ulcers. (See, e.g. PCT Publication WO84/01106for discussion of wound healing and related tissue repair).

A further aspect of the invention includes a therapeutic method andcomposition for repairing fractures and other conditions related to boneand/or cartilage defects or periodontal diseases. In addition, theinvention comprises therapeutic methods and compositions for woundhealing and tissue repair. Such compositions comprise a therapeuticallyeffective amount of at least one of the BMP-5 proteins of the inventionin admixture with a pharmaceutically acceptable vehicle, carrier ormatrix.

It is expected that the proteins of the invention may act in concertwith or perhaps synergistically with one another or with other relatedproteins and growth factors. Therapeutic methods and compositions of theinvention therefore comprise one or more of the proteins of the presentinvention. Further therapeutic methods and compositions of the inventiontherefore comprise a therapeutic amount of at least one protein of theinvention with a therapeutic amount of at least one of the other "BMP"proteins, BMP-1, BMP-2 (BMP-2A, BMP-2 Class I), BMP-3, BMP-4 (BMP-2B,BMP-2 Class II), BMP-6, and BMP-7 disclosed in co-owned and co-pendingU.S. applications described above. Such methods and compositions of theinvention may comprise proteins of the invention or portions thereof incombination with the above-mentioned "BMP" proteins or portions thereof.Such combination may comprise individual separate molecules from each ofthe proteins or heteromolecules such as heterodimers formed by portionsof the respective proteins. For example, a method and composition of theinvention may comprise a protein of the invention or a portion thereoflinked with a portion of a "BMP" protein to form a heteromolecule.

Further therapeutic methods and compositions of the invention comprisethe proteins of the invention or portions thereof in combination withother agents beneficial to the treatment of the bone and/or cartilagedefect, wound, or tissue in question. These agents include variousgrowth factors such as epidermal growth factor (EGF), fibroblast growthfactor (FGF), platelet derived growth factor (PDGF), transforming growthfactors (TGF-α and TGF-β), k-fibroblast growth factor (kFGF),parathyroid hormone (PTH), leukemia inhibitory factor (LIF/HILDA DIA)and insulin-like growth factors (IGF-I and IGF-II). Portions of theseagents may also be used in compositions of the invention.

The preparation and formulation of such physiologically acceptableprotein compositions, having due regard to pH, isotonicity, stabilityand the like, is within the skill of the art. The therapeuticcompositions are also presently valuable for veterinary applications dueto the apparent lack of species specificity in cartilage and bone growthfactor proteins. Domestic animals and thoroughbred horses in addition tohumans are desired patients for such treatment with the proteins of thepresent invention.

The therapeutic method includes administering the composition topically,systematically, or locally as an implant or device. When administered,the therapeutic composition for use in this invention is, of course, ina pyrogen-free, physiologically acceptable form. Further, thecomposition may desirably be encapsulated or injected in a viscous formfor delivery to the site of cartilage and/or bone or tissue damage.Topical administration may be suitable for wound healing and tissuerepair. Preferably for bone and/or cartilage formation, the compositionwould include a matrix capable of delivering the cartilage/bone proteinsof the invention to the site of bone and/or cartilage damage, providinga structure for the developing bone and cartilage and optimally capableof being resorbed into the body. The matrix may provide slow release ofthe BMP proteins or other factors comprising the composition. Suchmatrices may be formed of materials presently in use for other implantedmedical applications.

The choice of matrix material is based on biocompatibility,biodegradability, mechanical properties, cosmetic appearance andinterface properties. The particular application of the compositions ofthe invention will define the appropriate formulation. Potentialmatrices for the compositions may be biodegradable and chemicallydefined calcium sulfate, tricalciumphosphate, hydroxyapatite, polylacticacid and polyanhydrides. Other potential materials are biodegradable andbiologically well defined, such as bone or dermal collagen. Furthermatrices are comprised of pure proteins or extracellular matrixcomponents. Other potential matrices are nonbiodegradable and chemicallydefined, such as sintered hydroxyapatite, bioglass, aluminates, or otherceramics. Matrices may be comprised of combinations of any of the abovementioned types of material, such as polylactic acid and hydroxyapatiteor collagen and tricalciumphosphate. The bioceramics may be altered incomposition, such as in calcium-aluminate-phosphate and processing toalter pore size, particle size, particle shape, and biodegradability.

The dosage regimen will be determined by the attending physicianconsidering various factors which modify the action of the proteins ofthe invention. Factors which may modify the action of the proteins ofthe invention include the amount of bone weight desired to be formed,the site of bone damage, the condition of the damaged bone, the size ofa wound, type of damaged tissue, the patient's age, sex, and diet, theseverity of any infection, time of administration and other clinicalfactors. The dosage may vary with the type of matrix used in thereconstitution and the type or types of bone and/or cartilage proteinspresent in the composition. The addition of other known growth factors,such as EGF, PDGF, TGF-α, TGFβ, and IGF-I to the final composition, mayalso effect the dosage.

Progress can be monitored by periodic assessment of cartilage and/orbone growth and/or repair. The progress can be monitored, for example,using x-rays, histomorphometric determinations and tetracyclinelabeling.

The following examples illustrate practice of the present invention inrecovering and characterizing bovine cartilage and/or bone proteins ofthe invention and employing these proteins to recover the correspondinghuman protein or proteins and in expressing the proteins via recombinanttechniques.

EXAMPLE I Isolation of Bovine Cartilage/Bone Inductive Protein

Ground bovine bone powder (20-120 mesh, HELITREX) is prepared accordingto the procedures of M. R. Urist et al., Proc. Natl Acad. Sci USA,70:3511 (1973) with elimination of some extraction steps as identifiedbelow. Ten kgs of the ground powder is demineralized in successivechanges of 0.6N HCl at 4° C. over a 48 hour period with vigorousstirring. The resulting suspension is extracted for 16 hours at 4° C.with 50 liters of 2M CaCl₂ and 10 mM ethylenediamine-tetraacetic acid[EDTA], and followed by extraction for 4 hours in 50 liters of 0.5MEDTA. The residue is washed three times with distilled water before itsresuspension in 20 liters of 4M guanidine hydrochloride [GuCl], 20 mMTris (pH 7.4), 1 mM N-ethylmaleimide, 1 mM iodoacetamide, 1 mMphenylmethylsulfonyl fluorine as described in Clin. Orthop. Rel. Res.,171: 213 (1982). After 16 to 20 hours the supernatant is removed andreplaced with another 10 liters of GuCl buffer. The residue is extractedfor another 24 hours.

The crude GuCl extracts are combined, concentrated approximately 20times on a Pellicon apparatus with a 10,000 molecular weight cut-offmembrane, and then dialyzed in 50 mM Tris, 0.1M NaCl, 6M urea (pH 7.2),the starting buffer for the first column. After extensive dialysis theprotein is loaded on a 4 liter DEAE cellulose column and the unboundfractions are collected.

The unbound fractions are concentrated and dialyzed against 50 mM NaAc,50 mM NaCl (pH 4.6) in 6M urea. The unbound fractions are applied to acarboxymethyl cellulose column. Protein not bound to the column isremoved by extensive washing with starting buffer, and the materialcontaining protein having bone and/or cartilage formation activity asmeasured by the Rosen-modified Sampath - Reddi assay (described inExample III below) desorbed from the column by 50 mM NaAc, 0.25 mM NaCl,6M urea (pH 4.6). The protein from this step elution is concentrated 20-to 40-fold, then diluted 5 times with 80 mM KPO₄, 6M urea (pH 6.0). ThepH of the solution is adjusted to 6.0 with 500 mM K₂ HPO₄. The sample isapplied to an hydroxylapatite column (LKB) equilibrated in 80 mM KPO₄,6M urea (pH 6.0) and all unbound protein is removed by washing thecolumn with the same buffer. Protein having bone and/or cartilageformation activity is eluted with 100 mM KPO₄ (pH 7.4) and 6M urea.

The protein is concentrated approximately 10 times, and solid NaCl addedto a final concentration of 0.15M. This material is applied to aheparin - SEPHAROSE column equilibrated in 50 mM KPO₄, 150 mM NaCl, 6Murea (pH 7.4). After extensive washing of the column with startingbuffer, a protein with bone and/or cartilage inductive activity iseluted by 50 mM KPO₄, 700 mM NaCl, 6M urea (pH 7.4). This fraction isconcentrated to a minimum volume, and 0.4 ml aliquots are applied toSUPEROSE 6 and Superose 12 columns connected in series, equilibratedwith 4M GuCl, 20 mM Tris (pH 7.2) and the columns developed at a flowrate of 0.25 ml/min. The protein demonstrating bone and/or cartilageinductive activity corresponds to an approximate 30,000 dalton protein.

The above fractions from the SUPEROSE columns are pooled, dialyzedagainst 50 mM NaAc, 6M urea (pH 4.6), and applied to a PHARMACIA MONOSHR column. The column is developed with a gradient to 1.0M NaCl, 50 mMNaAc, 6M urea (pH 4.6). Active bone and/or cartilage formation fractionsare pooled. The material is applied to a 0.46×25 cm VYDAC C4 column in0.1% TFA and the column developed with a gradient to 90% acetonitrile,0.1% TFA (31.5% acetonitrile, 0.1% TFA to 49.5% acetonitrile, 0.1% TFAin 60 minutes at 1 ml per minute). Active material is eluted atapproximately 40-44% acetonitrile. Fractions were assayed for cartilageand/or bone formation activity. The active material from the C4 reversephase column is further fractionated on a MONOQ column. The protein isdialyzed against 6M urea, 25 mM diethanolamine, pH 8.6 and then appliedto a 0.5 by 5 cm MONOQ column (Pharmacia) which is developed with agradient of 6M urea, 25 mM diethanolamine, pH 8.6 and 0.5M NaCl, 6Murea, 25 mM diethanolamine, pH 8.6. Fractions are brought to pH 3.0 with10% trifluoroacetic acid (TFA).

Aliquots of the appropriate fractions are iodinated by one of thefollowing methods: P. J. McConahey et al, Int. Arch. Allergy, 29:185-189(1966); A. E. Bolton et al, Biochem J., 133:529 (1973); and D. F.Bowen-Pope, J. Biol. Chem., 237:5161 (1982). The iodinated proteinspresent in these fractions are analyzed by SDS gel electrophoresis.

EXAMPLE II Characterization of Bovine Cartilage/Bone Inductive Factor

A. Molecular Weight

Approximately 5 μg protein from Example I in 6M urea, 25 mMdiethanolamine, pH 8.6, approximately 0.3M NaCl is made 0.1% withrespect to SDS and dialyzed against 50 mM tris/HCl 0.1% SDS pH 7.5 for16 hrs. The dialyzed material is then electrophorectically concentratedagainst a dialysis membrane [Hunkapillar et al Meth. Enzymol. 91:227-236 (1983)] with a small amount of I 125 labelled counterpart. Thismaterial (volume approximately 100 μl) is loaded onto a 12%polyacrylamide gel and subjected to SDS-PAGE [Laemmli, U.K. Nature,227:680-685 (1970)] without reducing the sample with dithiothreitol. Themolecular weight is determined relative to prestained molecular weightstandards (Bethesda Research Labs). Following autoradiography of theunfixed gel the approximate 28,000-30,000 dalton band is excised and theprotein electrophoretically eluted from the gel (Hunkapillar et alsupra). Based on similar purified bone fractions as described in theco-pending "BMP" applications described above wherein bone and/orcartilage activity is found in the 28,000-30,000 region, it is inferredthat this band comprises bone and/or cartilage inductive fractions.

B. Subunit Characterization

The subunit composition of the isolated bovine bone protein is alsodetermined. The eluted protein described above is fully reduced andalkylated in 2% SDS using iodoacetate and standard procedures andreconcentrated by electrophoretic packing. The fully reduced andalkylated sample is then further submitted to SDS-PAGE on a 12% gel andthe resulting approximate 14,000-20,000 dalton region having a doubletappearance located by autoradiography of the unfixed gel. A faint bandremains at the 28,000-30,000 region. Thus the 28,000-30,000 daltonprotein yields a broad region of 14,000-20,000 which may otherwise alsobe interpreted and described as comprising two broad bands ofapproximately 14,000-16,000 and 16,000-20,000 daltons.

EXAMPLE III Rosen Modified Sampath-Reddi Assay

A modified version of the rat bone formation assay described in Sampathand Reddi, Proc. Natl. Acad. Sci. U.S.A., 80:6591-6595 (1983) is used toevaluate bone and/or cartilage activity of the proteins of theinvention. This modified assay is herein called the Rosen-modifiedSampath-Reddi assay. The ethanol precipitation step of the Sampath-Reddiprocedure is replaced by dialyzing (if the composition is a solution) ordiafiltering (if the composition is a suspension) the fraction to beassayed against water. The solution or suspension is then redissolved in0.1% TFA, and the resulting solution added to 20 mg of rat matrix. Amock rat matrix sample not treated with the protein serves as a control.This material is frozen and lyophilized and the resulting powderenclosed in #5 gelatin capsules. The capsules are implantedsubcutaneously in the abdominal thoracic area of 21-49 day old male LongEvans rats. The implants are removed after 5-21 days. Half of eachimplant is used for alkaline phosphatase analysis [See, A. H. Reddi etal., Proc. Natl Acad Sci., 69:1601 (1972)].

The other half of each implant is fixed and processed for histologicalanalysis. Glycolmethacrylate sections (1 μm) are stained with Von Kossaand acid fuschin or toluidine blue to score the amount of induced boneand cartilage formation present in each implant. The terms +1 through +5represent the area of each histological section of an implant occupiedby new bone and/or cartilage cells and newly formed bone and matrix. Twoscoring methods are herein described. The first describes the originalscoring method while the second describes the later adopted scoringmethod. A score of +5 indicates that greater than 50% of the implant isnew bone and/or cartilage produced as a direct result of protein in theimplant. A score of +4, +3, +2 and +1 would indicate that greater than40%, 30%, 20% and 10% respectively of the implant contains new cartilageand/or bone. The scoring method later adopted (which hereinafter may bereferred to as the "modified" scoring method) is as follows: Threenon-adjacent sections are evaluated from each implant and averaged."+/-" indicates tentative indentification of cartilage or bone; "+1"indicates >10% of each section being new cartilage or bone; "+2", >25%;"+3", >50%; "+4", ˜75%; "+5", >80%. A "-" indicates that the implant isnot recovered. The scores of the individual implants are tabulated toindicate assay variability.

It is contemplated that the dose response nature of the cartilage and/orbone inductive protein containing samples of the matrix samples willdemonstrate that the amount of bone and/or cartilage formed increaseswith the amount of cartilage/bone inductive protein in the sample. It iscontemplated that the control samples will not result in any bone and/orcartilage formation.

As with other cartilage and/or bone inductive proteins such as theabove-mentioned "BMP" proteins, the bone and/or cartilage formed isexpected to be physically confined to the space occupied by the matrix.Samples are also analyzed by SDS gel electrophoresis and isoelectricfocusing followed by autoradiography. The activity is correlated withthe protein bands and pI. To estimate the purity of the protein in aparticular fraction an extinction coefficient of 1 OD/mg-cm is used asan estimate for protein and the protein is run on SDS-PAGE followed bysilver staining or radioiodination and autoradiography.

EXAMPLE IV Bovine BMP-5 Protein Composition

The gel slice of the approximate 14,000-20,000 dalton region describedin Example IIB is fixed with methanol-acetic acid-water using standardprocedures, briefly rinsed with water, then neutralized with 0.1Mammonium bicarbonate. Following dicing the gel slice with a razor blade,the protein is digested from the gel matrix by adding 0.2 μg ofTPCK-treated trypsin (Worthington) and incubating the gel for 16 hr. at37 degrees centigrade. The resultant digest is then subjected to RPHPLCusing a C4 VYDAC RPHPLC column and 0.1% TFA-water 0.1% TFAwater-acetonitrile gradient. The resultant peptide peaks were monitoredby UV absorbance at 214 and 280 nm and subjected to direct aminoterminal amino acid sequence analysis using an Applied Biosystems gasphase sequenator (Model 470A). One tryptic fragment is isolated bystandard procedures having the following amino acid sequence asrepresented by the amino acid standard three-letter symbols and where"Xaa" indicates an unknown amino acid the amino acid in parenthesesindicates uncertainty in the sequence:

    Xaa-His-Glu-Leu-Tyr-Val-Ser-Phe-(Ser)

The following four oligonucleotide probes are designed on the basis ofthe amino acid sequence of the above-identified tryptic fragment andsynthesized on an automated DNA synthesizer.

PROBE #1: GTRCTYGANATRCANTC

PROBE #2: GTRCTYGANATRCANAG

PROBE #3: GTRCTYAAYATRCANTC

PROBE #4: GTRCTYAAYATRCANAG

The standard nucleotide symbols in the above identified probes are asfollows: A,adenine; C,cytosine; G,guanine; T,thymine; N, adenine orcytosine or quanine or thymine; R,adenine or quanine; and Y,cytosine orthymine.

Each of the probes consists of pools of oligonucleotides. Because thegenetic code is degenerate (more than one codon can code for the sameamino acid), a mixture of oligonucleotides is synthesized that containsall possible nucleotide sequences encoding the amino acid sequence ofthe tryptic. These probes are radioactively labeled and employed toscreen a bovine cDNA library as described below.

Poly(A) containing RNA is isolated by oligo(dT) cellulose chromatographyfrom total RNA isolated from fetal bovine bone cells by the method ofGehron-Robey et al in Current Advances in Skeletogenesis, ElsevierScience Publishers (1985). The total RNA was obtained from Dr. MarionYoung, National Institute of Dental Research, National Institutes ofHealth. A cDNA library is made in lambda gt10 (Toole et al supra) andplated on 50 plates at 8000 recombinants per plate. These recombinants(400,000) are screened on duplicate nitrocellulose filters with acombination of Probes 1, 2, 3, and 4 using the Tetramethylammoniumchloride (TMAC) hybridization procedure [see Wozney et al Science, 242:1528-1534 (1988)]. Twenty-eight positives are obtained and are replatedfor secondaries. Duplicate nitrocellulose replicas again are made. Oneset of filters are screened with Probes 1 and 2; the other with Probes 3and 4. Six positives are obtained on the former, 21 positives with thelatter. One of the six, called HEL5, is plague purified, a phage platestock made, and bacteriophage DNA isolated. This DNA is digested withEcoRI and subcloned into M13 and pSP65 (Promega Biotec, Madison, Wis.)[Melton, et al., Nucl. Acids Res. 12:7035-7056 (1984)]. The DNA sequenceand derived amino acid sequence of this fragment is shown in Table I.

DNA sequence analysis of this fragment in M13 indicates that it encodesthe desired tryptic peptide sequence set forth above, and this derivedamino acid sequence is preceded by a basic residue (Lys) as predicted bythe specificity of trypsin. The underlined portion of the sequence inTable I from amino acid #42 to #48 corresponds to the tryptic fragmentidentified above from which the oligonucleotide probes are designed. Thederived amino acid sequence Ser-Gly-Ser-His-Gln-Asp-Ser-Ser-Arg as setforth in Table I from amino acid #15 to #23 is noted to be similar to atryptic fragment sequence Ser-Thr-Pro-Ala-Gln-Asp-Val-Ser-Arg found inthe 28,000-30,000 dalton purified bone preparation as described in the"BMP" co-pending applications mentioned above. This fragment set forthin Table I is a portion of the DNA sequence which encodes a bovine BMP-5protein of the invention. The DNA sequence indicates an open readingframe from the 5' end of the clone of 420 base pairs, encoding a partialpeptide of 140 amino acid residues (the first 7 nucleotides are of theadaptors used in the cloning procedure). An in-frame stop codon (TAA)indicates that this clone encodes the carboxy-terminal part of thebovine BMP-5 cartilage/bone protein of the invention.

                                      TABLE I                                     __________________________________________________________________________     ##STR1##                                                                      ##STR2##                                                                      ##STR3##                                                                      ##STR4##                                                                      ##STR5##                                                                      ##STR6##                                                                      ##STR7##                                                                      ##STR8##                                                                     481CTACAATAAAAAATATCTTTCGGATAAAAGGGGAATTTAATAAAATTAGTCTGGCTCATT540            541TCATCTCTGTAACCTATGTACAAGAGCATGTATATAGT578                                  __________________________________________________________________________

The remaining positive clones isolated with probes #1, #2, #3, and #4described above are screened with HEL5 and a further clone is identifiedthat hybridizes under reduced hybridization conditions [5×SSC, 0.1% SDS,5×Denhardt's, 100 lg/ml salmon sperm DNA standard hybridization buffer(SHB) at 65#C, wash in 2×SSC 0.1% SDS at 65#C]. This clone is plaquepurified, a phage plate stock made and bacteriophage DNA isolated. TheDNA sequence and derived amino acid sequence of a portion of this cloneis shown in Table II. This sequence represents the DNA sequence encodinga BMP-6 cartilage/bone protein.

The first underlined portion of the sequence in Table II from amino acid#97-amino acid #105 corresponds to the tryptic fragment found in the28,000-30,000 dalton purified bovine bone preparation (and its reducedformat approximately 18,000-20,000 dalton reduced form) as described inthe "BMP" co-pending applications mentioned above. The second underlinedsequence in Table II from amino acid #124-amino acid #130 corresponds tothe tryptic fragment identified above from which the oligonucleotideprobes are designed.

The DNA sequence of Table II indicates an open reading frame of 666 basepairs starting from the 5' end of the sequence of Table II, encoding apartial peptide of 222 amino acid residues. An in-frame stop codon (TGA)indicates that this clone encodes the carboxy-terminal part of a bovineBMP-6 protein of the invention. Based on knowledge of other BMP proteinsand other proteins in the TGF-b family, it is predicted that theprecursor polypeptide would be cleaved at the three basic residues(ArgArgArg) to yield a mature peptide beginning with residue 90 or 91 ofthe sequence of Table II.

                                      TABLE II                                    __________________________________________________________________________     ##STR9##                                                                      ##STR10##                                                                     ##STR11##                                                                     ##STR12##                                                                     ##STR13##                                                                     ##STR14##                                                                     ##STR15##                                                                     ##STR16##                                                                     ##STR17##                                                                     ##STR18##                                                                     ##STR19##                                                                     ##STR20##                                                                     ##STR21##                                                                     ##STR22##                                                                     ##STR23##                                                                     ##STR24##                                                                     ##STR25##                                                                    __________________________________________________________________________

EXAMPLE V Human BMP-5 Proteins

Human cell lines which synthesize BMP-5 and/or BMP-6 mRNAs areidentified in the following manner. RNA is isolated from a variety ofhuman cell lines, selected for poly(A)-containing RNA by chromatographyon oligo(dT) cellulose, electrophoresed on a formaldehyde-agarose gel,and transferred to nitrocellulose. A nitrocellulose replica of the gelis hybridized to a single stranded M13 ³² P-labeled probe correspondingto the above mentioned BMP-5 EcoRI-BglII fragment containing nucleotides1-465 of the sequence of Table I. A strongly hybridizing band isdetected in the lane corresponding to the human osteosarcoma cell lineU-2OS RNA. Another nitrocellulose replica is hybridized to a singlestranded M13 ³² P-labeled probe containing the PstI-SmaI fragment ofbovine BMP-6 (corresponding to nucleotides 106-261 of Table II). It isfound that several RNA species in the lane corresponding to U-2OS RNAhybridize to this probe.

A cDNA library is made in the vector lambda ZAP (Stratagene) from U-2OSpoly(A)-containing RNA using established techniques (Toole et al.).750,000 recombinants of this library are plated and duplicatenitrocellulose replicas made. The SmaI fragment of bovine BMP-6corresponding to nucleotides 259-751 of Table II is labeled bynick-translation and hybridized to both sets of filters in SHB at 65°.One set of filters is washed under stringent conditions (0.2×SSC, 0.1%SDS at 65°), the other under reduced stringency conditions (1×SSC, 0.1%SDS at 65°). Many duplicate hybridizing recombinants (approximately 162)are noted. 24 are picked and replated for secondaries. Threenitrocellulose replicas are made of each plate. One is hybridized to theBMP-6 SmaI probe, one to a nick-translated BMP-6 PstI-SacI fragment(nucleotides 106-378 of Table II), and the third to the nick-translatedBMP-5 XbaI fragments (nucleotides 1-76 of Table I). Hybridization andwashes are carried out under stringent conditions.

17 clones that hybridized to the third probe more strongly than to thesecond probe are plaque purified. DNA sequence analysis of one of these,U2-16, indicated that it encodes human BMP-5. U2-16 was deposited withthe American Type Culture Collection (ATCC), Rockville, Md. on Jun. 22,1989 under accession number ATCC 68019. U2-16 contains an insert ofapproximately 2.1 Kb. The DNA sequence and derived amino acid sequenceof U2-16 is shown below in Table III. This clone is expected to containall of the nucleotide sequence necessary to encode the BMP-5 proteins.The cDNA sequence of Table III contains an open reading frame of 1362bp, encoding a protein of 454 amino acids, preceded by a 5' untranslatedregion of 700 bp with stop codons in all frames, and contains a 3'untranslated region of 90 bp following the in frame stop codon (TAA).

This protein of 454 amino acids has a molecular weight of approximately52,000 kd as predicted by its amino acid sequence, and is contemplatedto represent the primary translation product. Based on knowledge ofother BMP proteins and other proteins within the TGF-b family, cleavageof the precursor polypeptide may occur after the tribasic peptide LysArg Lys yielding a 132 amino acid mature peptide beginning with aminoacid #323 "Asn". However, the presence of di- or tribasic amino acidsequence is not an absolute requirement for proteolytic processing, as anumber of prohormones are known to be processed after single arginineswhich conform to a consensus cleavage sequence arginine-X-X-arginine. Itis therefore contemplated that the precursor polypeptide isproteolytically processed after the Arg-Ser-Val-Arg sequence yielding apolypeptide comprising 138 amino acids from amino acid #317 (Ala) to#454 (His) as shown in Table III with a calculated molecular weight of15.6 kD. The processing of BMP-5 into the mature form is expected toinvolve dimerization and removal of the N-terminal region in a manneranalogous to the processing of the related protein TGF-β [L. E. Gentry,et al., Molec. & Cell. Biol. 8:4162 (1988); R. Dernyck, et al., Nature316:701 (1985)].

It is contemplated therefore that the mature active species of BMP-5comprises a homodimer of 2 polypeptide subunits each subunit comprisingamino acid #323-#454 with a predicted molecular weight of approximately15,000 daltons. Further active BMP-5 species are contemplated, forexample, proprotein dimers or proprotein subunits linked to maturesubunits. Additional active species may comprise amino acid #329-#454such species including homologous the tryptic sequences found in thepurified bovine material. Also contemplated are BMP-5 proteinscomprising amino acids #353-#454 thereby including the first conservedcysteine residue

The underlined sequence of Table III from amino acid #329 to #337Ser-Ser-Ser-His-Gln-Asp-Ser-Ser-Arg shares homology with the bovinesequence of Table I from amino acid #15 to #23 as discussed above inExample IV. Each of these sequences shares homology with a trypticfragment sequence Ser-Thr-Pro-Ala-Gln-Asp-Val-Ser-Arg found in the28,000-30,000 dalton purified bone preparation (and its reduced form atapproximately 18,000-20,000 daltons) as described in the "BMP"co-pending applications mentioned above.

The underlined sequence of Table III from amino acid #356 to #362His-Glu-Leu-Tyr-Val-Ser-Phe corresponds to the tryptic fragmentidentified in the bovine bone preparation described above from which theoligonucleotide probes are designed.

                                      TABLE III                                   __________________________________________________________________________    Human                                                                         __________________________________________________________________________     ##STR26##                                                                     ##STR27##                                                                     ##STR28##                                                                     ##STR29##                                                                     ##STR30##                                                                     ##STR31##                                                                     ##STR32##                                                                     ##STR33##                                                                     ##STR34##                                                                     ##STR35##                                                                     ##STR36##                                                                     ##STR37##                                                                     ##STR38##                                                                     ##STR39##                                                                    __________________________________________________________________________

                                      TABLE III(a)                                __________________________________________________________________________     ##STR40##                                                                     ##STR41##                                                                     ##STR42##                                                                     ##STR43##                                                                     ##STR44##                                                                     ##STR45##                                                                     ##STR46##                                                                     ##STR47##                                                                     ##STR48##                                                                     ##STR49##                                                                     ##STR50##                                                                     ##STR51##                                                                    __________________________________________________________________________

                                      TABLE III(b)                                __________________________________________________________________________     ##STR52##                                                                     ##STR53##                                                                     ##STR54##                                                                     ##STR55##                                                                     ##STR56##                                                                     ##STR57##                                                                     ##STR58##                                                                     ##STR59##                                                                     ##STR60##                                                                     ##STR61##                                                                     ##STR62##                                                                    __________________________________________________________________________

                                      TABLE III(c)                                __________________________________________________________________________     ##STR63##                                                                     ##STR64##                                                                     ##STR65##                                                                     ##STR66##                                                                     ##STR67##                                                                     ##STR68##                                                                     ##STR69##                                                                     ##STR70##                                                                     ##STR71##                                                                    __________________________________________________________________________

The invention encompasses the corresponding bovine and human BMP-5genomic sequences. These genes can be isolated using the cDNA sequencesset forth in Table I and Table III as probes to screen genomic librariesusing techniques known to those skilled in the art.

When the tryptic sequence His-Glu-Leu-Tyr-Val-Ser-Phe-(Ser) describedabove was identified, it was noted to be similar to the sequenceHis-Pro-Leu-Tyr-Val-Asp-Phe-Ser found in the bovine and humancartilage/bone protein BMP-2A sequence, for instance as described inco-pending U.S. application Ser. No. 179,100. Human BMP-5 shareshomology with other BMP molecules as well as other members of the TGF-bsuperfamily of molecules. The cysteine-rich carboxy-terminal 102 aminoacids residues of human BMP-5 shares, the following homologies with BMPproteins disclosed in copending applications described above: 61%identity with BMP-2; 43% identity with BMP-3, 59% identity with BMP-4;91% identity with BMP-6; and 88% identity with BMP-7. Human BMP-5further shares the following homologies: 38% identity with TGF-β3; 37%identity with TGF-b2; 36% identity with TGF-b1; 25% identity withMullerian Inhibiting Substance (MIS), a testicular glycoprotein thatcauses regression of the Mullerian duct during development of the maleembryo; 25% identity with inhibin a; 38% identity with inhibin b_(B) ;45% identity with inhibin b_(A) ; 56% identity with Vgl, a Xenopusfactor which may be involved in mesoderm induction in earlyembryogenesis (Lyons, et al., PNAS USA 86:4554-4558 (1989)]; and 57%identity with Dpp the product of the Drosophila decapentaplegic locuswhich is required for dorsal-ventral specification in earlyembryogenesis and is involved in various other developmental processesat later stages of development [Padgett, et al., Nature 325:81-84(1987)].

The procedures described above and additional methods known to thoseskilled in the art may be employed to isolate other related proteins ofinterest by utilizing the bovine or human proteins as a probe source.Such other proteins may find similar utility in, inter alia, fracturerepair, wound healing and tissue repair.

EXAMPLE VI Expression of the BMP-5 Proteins

In order to produce bovine, human or other mammalian proteins of theinvention, the DNA encoding it is transferred into an appropriateexpression vector and introduced into mammalian cells or other preferredeukaryotic or prokaryotic hosts by conventional genetic engineeringtechniques. It is contemplated that the preferred expression system forbiologically active recombinant human proteins of the invention may bestably transformed mammalian cells. For transient expression, the cellline of choice is SV40 transformed African green monkey kidney COS-1 orCOS-7 which typically produce moderate amounts of the protein encodedwithin the plasmid for a period of 1-4 days. For stable high levelexpression, it is further contemplated that the preferred mammaliancells will be Chinese hamster ovary (CHO) cells.

The transformed host cells are cultured and the BMP-5 protein expressedthereby is recovered, isolated and purified. Characterization ofexpressed proteins is carried out using standard techiques. For example,characterization may include pulse labeling with [³ 5^(S) ] methionineor cysteine and analysis by polyacrylamide electrphoresisTherecombinantly expressed BMP-5 proteins are free of proteinaceousmaterials with which they are co-produced and with which they ordinarilyare associated in nature, as well as from other contaminants, such asmaterials found in the culture media.

In order to express biologically active human BMP-5 a selected host cellis transformed, using techniques known to those skilled in the art ofgenetic engineering, with a DNA sequence encoding human BMP-5 protein.The DNA comprises the nucleotide sequence from nucleotide #1665 to #2060set forth in Table III encoding amino acid #323 to #454. The DNA maycomprise the DNA sequence from nucleotide #699 to #2060 set forth inTable III. The transformed host cells are cultured and the BMP-5 proteincomprising the amino acid sequence from amino acid #323 to amino acid#454 set forth in Table III is expressed. The expressed protein isrecovered, isolated and purified from the culture and culture medium.The purified protein is substantially free from other proteinaceousmaterials with which it is co-produced, and from other contaminants.

A. Vector Construction

As described above, numerous expression vectors known in the art may beutilized in the expression of BMP proteins of the invention. The vectorutilized in the following examples are pMT21, a derivitive of pMT₂, andpEMC2β1 derived from pMT21 though other vectors may be suitable inpractice of the invention.

pMT₂ is derived from pMT2-VWF, which has been deposited with theAmerican Type Culture Collection (ATCC), Rockville, Md. (USA) underaccession number ATCC 67122 under the provisions of the Budapest Treaty.EcoRI digestion excises the cDNA insert present in pMT-VWF, yieldingpMT2 in linear form which can be ligated and used to transform E. coliHB 101 or DH-5 to ampicillin resistance. Plasmid pMT2 DNA can beprepared by conventional methods.

pMT21 is then constructed using loopout/in mutagenesis [Morinaga, etal., Biotechnology 84:636 (1984)]. This removes bases 1075 to 1170(inclusive). In addition it inserts the following sequence: 5' TCGA 3'.This sequence completes a new restriction site, XhoI. This plasmid nowcontains 3 unique cloning sites PstI, EcoRI, and XhoI.

In addition, pMT21 is digested with EcoRV and XhoI, treating thedigested DNA with Klenow fragment of DNA polymerase I and ligating ClaIlinkers (NEBio Labs, CATCGATG). This removes bases 2171 to 2420 startingfrom the HindIII site near the SV40 origin of replication and enhancersequences of pMT2 and introduces a unique Cla I site, but leaves theadenovirus VAI gene intact.

pEMC2β1 is derived from pMT21. pMT21 is cut with EcoRI and XhoI whichcleaves the plasmid two adjacent cloning sites situated after the IgGintron. An EMCV fragment of 508 base pairs is cut from pMT₂ ECAT₁ [S. K.Jong, et al., J. Virol. 63:1651-1660 (1989)] with the restrictionenzymes EcoRI and TagαI. A pair of oligonucleotides 68 nucleotides cgaggttaaaaaa cgtctaggcc ccccgaacca cggggacgtg gttttccttt gaaaaacacg attgcin length are synthesized to duplicate the EMCV sequence up to the ATG.The ATG is changed to an ATT, and a C is added, creating a XhoI site atthe 3' end. A tagαI site is situated at the 5' end. Ligation of the MT21EcoRI to XhoI fragment to the EMCV EcoRI to TagαI fragment and to theTagαI/XhoI oligonucleotides produces the vector EMC@B1. This vectorcontains the SV40 origin of replication and enhancer, the adenovirusmajor late promoter, a cDNA copy of the majority of the adenovirustripartite leader sequence, a small hybrid intervening sequence, an SV40polyadenylation signal and the dadenovirus VA I gene, DHFR andB-lactamase markers and an EMC sequence, in appropriate relationships todirect the high level expression of the desired cDNA in mammalian cells.

B. BMP-5 Vector Construction

A derivative of the BMP-5 cDNA sequence set forth in Table IIIcomprising the the nucleotide sequence from nucleotide #699 to #2070 isspecifically amplified. The oligonucleotidesCGACCTGCAGCCACCATGCATCTGACTGTA and TGCCTGCAGTTTAATATTAGTGGCAGC areutilized as primers to allow the amplification of nucleotide sequence#699 to #2070 of Table III from the insert of clone U2-16 describedabove in Example V. This procedure introduces the nucleotide sequenceCGACCTGCAGCCACC immediately preceeding nucleotide #699 and thenucleotide sequence CTGCAGGCA immediately following nucleotide #2070.The addition of these sequences results in the creation of PstIrestriction endonuclease recognition sites at both ends of the amplifiedDNA fragment. The resulting amplified DNA product of this procedure isdigested with the restriction endonuclease PstI and subcloned into thePstI site of the pMT2 derivative pMT21 described above. The resultingclone is designated H5/5/pMT.

The insert of H5/5/pMT is excised by PstI digestion and subcloned intothe plasmid vector pSP65 at the PstI site resulting in BMP5/SP6.BMP5/SP6 and U2-16 are digested with the restriction endonucleases NsiIand NdeI to excise the portion of their inserts corresponding tonucleotides #704 to #1876 of Table III. The resulting 1173 nucleotideNsiI-Ndei fragment of clone U2-16 is ligated into the NsiI-NdeI site ofBMP5/SP6 from which the corresponding 1173 nucleotide NsiI-NdeI fragmenthad been removed. The resulting clone is designated BMP5mix/SP64.

Direct DNA sequence analysis of BMP5mix/SP64 is performed to confirmidentity of the nucleotide sequences produced by the amplification tothose set forth in Table III. The clone BMP5mix/SP64 is digested withthe restriction endonuclease PstI resulting in the excision of an insertcomprising the nucleotides #699 to #2070 of Table III and the additionalsequences containing the PstI recognition sites as described above. Theresulting 1382 nucleotide PstI fragment is subcloned into the PstI siteof the pMT2 derivative pMT21 and pEMC2β1. These clones are designatedBMP5mix/pMT21#2 and BMp5mix/EMC#11.

EXAMPLE VII Transient COS Cell Expression

To obtain transient expression of BMP-5 proteins a vector containing thecDNA for BMP-5, BMP5mix/pMT21#2, is transfected into COS-1 cells usingthe electroporation method. Other suitable transfection methods includeDEAE-dextran, and lipofection. Approximately 48 hours later, cells areanalysed for expression of both intracellular and secreted BMP-5 proteinby metabolic labelling with [³⁵ S] methionine and polyacrylamide gelelectrophoresis. Intracellular BMP is analyzed in cells which aretreated with tunicamycin, an inhibitor of N-linked glycosylation. Intunicamycin-treated cells, the nonglycosylated primaryl translationproduct migrates as a homogeneous band of predictable size and is ofteneasier to discern in polyacrylamide gels than the glycosylated form ofthe protein. In each case, intracelluar protein in tunicamycin-treatedcells is compared to a duplicate plate of transfected, but untreatedCOS-1 cells.

The results demonstrate that intracellular forms of BMP-5 ofapproximately 52 Kd and 57 Kd are made by COS cells. The 52 Kd proteinis the size predicted by the primary sequence of the the BMP-5 cDNAclone. Following treatment of the cells with tunicamycin, only the 52 Kdform of BMP-5 is made, suggesting that the 57 Kd protein is aglycosylated derivative of the 52 Kd primary translation product. The 57Kd protein is secreted into the conditioned medium and is apparently notefficiently processed by COS-1 cells into the pro and mature peptides.

EXAMPLE VIII CHO Cell Expression

DHFR deficient CHO cells (DUKX B11) are transfected by electroporationwith BMP-5 expression vectors described above, and selected forexpression of DHFR by growth in nucleoside-free media. Other methods oftransfection, including but not limited to CaPO₄ precipitation,protoplast fusion, microinjection, and lipofection, may also beemployed. In order to obtain higher levels of expression moreexpediently, cells may be selected in nucleoside-free media supplementedwith 5 nM, 20 nM or 100 nM MTX. Since the DHFR selectable marker isphysically linked to the BMP-5 cDNA as the second gene of a bicistroniccoding region, cells which express DHFR should also express the BMP-5encoded within the upstream cistron. Either single clones, or pools ofcombined clones, are expanded and analyzed for expression of BMPprotein. Cells are selected in stepwise increasing concentrations of MTX(5 nM, 20 nM, 100 nM, 500 nM, 2 uM, 10 uM, and 100 uM) in order toobtain cell lines which contain multiple copies of the expression vectorDNA by virtue of gene amplification, and hence secrete large amounts ofBMP-5 protein.

Using standard techniques cell lines are screened for expression ofBMP-5 RNA, protein or activity, and high expressing cell lines arecloned or recloned at the appropriate level of selection to obtain amore homogeneous population of cells. The resultant cell line is thenfurther characterized for BMP-5 DNA sequences, and expression of BMP-5RNA and protein. Suitable cell lines can then be used for producingrecombinant BMP protein.

The BMP-5 vector BMP5mix/pMT21#2 and BMP5mix/EMC#11 described above aretransfected into CHO cells by electroporation, and cells are selectedfor expression of DHFR in nucleoside free medium. Clonal cell lines areobtained from individual colonies and are subsequently selected stepwisefor resistence to MTX, and are analyzed for secretion of BMP-5 proteins.In some cases cell lines may be maintained as pools and cloned at laterstages of MTX selection. One particular cell line further described, isdesignated 5E10 is sequentially selected for resistence to 0.02 uM, 0.1mM, 0.5 uM and 2.0 uM MTX to obtain amplified expression of BMP-5.

The amount of BMP-5 recovered in conditioned medium from 5E10 and othercell lines that express BMP-5 can be increased by including heparin,suramin, dextran sulfate, pectic acid, sodium sulfate, or relatedcompounds in the growth medium.

As described in Example V. the cDNA for BMP-5 encodes a protein ofapproximately 52 kD. Following processing within the cell that includes,but may not be limited to, propeptide cleavage, glycosylation, and dimeror multimer formation, multiple BMP-5 peptides are produced. There areat least 4 candidate peptides for processed forms of the BMP-5 proteindiscernable following SDS PAGE under reducing conditions; a peptide ofapproximately 65 kD, a peptide of approximately 35 kD, and a doublet ofapproximately 22 kD molecular weight. Other less abundant BMP-5 peptidesmay also be present. By comparison to the processing of other relatedBMP molecules and the related protein TGF-beta, the 65 Kd protein likelyrepresents unprocessed BMP-5, the 35 Kd species represents thepropeptide, and the 22 Kd doublet represents the mature peptide.

Material from a BMP-5 cell line is analyzed in a 2-dimensional gelsystem. In the first dimension, proteins are electrophoresed undernonreducing conditions. The material is then reduced, andelectrophoresed in a second polyacrylamide gel. Proteins that formdisulfide-bonded dimers or multimers will run below a diagonal acrossthe second reduced gel. Results from analysis of BMP-5 protein indicatesthat a significant amount of the mature BMP-5 peptides can formhomodimers of approximately 30-35 kD that reduce to the 22 kD doubletobserved in one dimensional reduced gels. A fraction of the maturepeptides are apparently in a disulfide-bonded complex with the propeptide. The amount of this complex is minor relative to the maturehomodimer. In addition, some of the unprocessed protein can apparantlyform homodimers or homomultimers.

EXAMPLE IX Purification and Biological Activity of Expressed BMP-5Proteins

To measure the biological activity of the expressed BMP-5 proteinsobtained in Example VIII above, the BMP-5 proteins are recovered fromthe culture media and purified by isolating them from otherproteinaceous materials with which they are co-produced, as well as fromother contaminants. BMP-5 proteins may be partially purified on aHeparin SEPHAROSE column and further purified using standardpurification techniques known to those skilled in the art. The BMP-5protein is mixed with 20 mg of rat matrix and then assayed for in vivobone and/or cartilage formation activity by the Rosen-modified Sampath -Reddi assay. A mock transfection supernatant fractionation is used as acontrol.

The implants containing rat matrix to which specific amounts of humanBMP-5 proteins of the invention have been added are removed from ratsafter approximately seven days and processed for histologicalevaluation. Representative sections from each implant are stained forthe presence of new bone mineral with von Kossa and acid fuschin, andfor the presence of cartilage-specific matrix formation using toluidineblue. The types of cells present within the section, as well as theextent to which these cells display phenotype are evaluated and scoredas described in Example III.

A. Purification of BMP-5 Proteins

(1) As one example of BMP-5 purification 4 ml of the collected posttransfection conditioned medium supernatant from one 100 mm culture dishis concentrated approximately 10 fold by ultrafiltration on a YM 10membrane and then dialyzed against 20 mM Tris, 0.15M NaCl, pH 7.4(starting buffer). This material is then applied to a 1.1 ml HeparinSEPHAROSE column in starting buffer. Unbound proteins are removed by an8 ml wash of starting buffer, and bound proteins, including proteins ofthe invention, are desorbed by a 3-4 ml wash of 20 mM Tris, 2.0M NaCl,pH 7.4.

The proteins bound by the Heparin column are concentrated approximately10-fold on a Centricon 10 and the salt reduced by diafiltration with0.1% trifluoroacetic acid.

Further purification may be achieved by preparative NaDodSO₄ /PAGE[Laemmli, Nature 227:680-685 (1970)]. For instance, approximately 300 μgof protein is applied to a 1.5 mm-thick 12.5% gel: recovery is estimatedby adding L[³⁵ S]methionine-labeled BMP protein purified overheparin-Sepharose as described above. Protein may be visualized bycopper staining of an adjacent lane [Lee, et al., Anal. Biochem.166:308-312 (1987)]. Appropriate bands are excised and extracted in 0.1%NaDodSO₄ /20 mM Tris, pH 8.0. The supernatant may be acidified with 10%CF₃ COOH to pH 3 and the proteins are desalted on 5.0×0.46 cm Vydac C₄column (The Separations Group, Hesperia, Calif.) developed with agradient of 0.1% CF₃ COOH to 90% acetonitrile/0.1% CFF₃ COOH.

(2) In another example, soluble heparin (100 ug/ml) is removed from ofBMP-5 protein in conditioned media from 5E10(2) 2.0MTX (described above)using butyl TSK hydrophobic interactive chrmatography (HIC). Theconditioned media is brought to 2M NaCl by addition of solid NaCl. Theconditioned media is then loaded on butyl TSK equilabrated in 2M NaCl,50 mM Tris, pH 7.4 washed with 0M NaCl, 50 mM Tris, Ph 7.4, followed byelution with 1% Np-40, 6M urea, 50 mM Tris, pH 7.4 resulting inapproximately 98% removal of soluble heparin.

The resulting material is then subjected to heparin SEPHAROSEchromatography. The material is directly loaded onto a heparin columnequilabrated in 50 mM Tris, 6M urea, 0M NaCl, washed and eluted with agradient of 0-2M NaCl. This material is analyzed by western blot and theBMP-5 containing fractions (0.3-0.8M NaCl) are pooled. The antibody isdirected against the C-terminal presumed mature portion. Proteins of35-40 kD non-reduced, 20-22 kD reduced, and higher molecular weightdimers are observed.

The BMP-5 containing fractions are concentrated and diafiltered to bringthe sample to 0.1% TFA loaded onto a reverse phase column and elutedwith a gradient from 30% to 60% B (A=0.1% TFA; B=0.1% TFA in 90%acetonitrile) in 75 min at 1 ml/min. SDS-PAGE analysis reveals severalmolecular weight species of BMP-5 proteins which are further describedbelow. The mature species which is contemplated to comprise a homodimerof amino acids #317-#454 as shown in Table III comprises approximately46-49% of the resulting molecular weight species.

B. Characterization of BMP-5 Proteins

One dimensional Western blot analysis reveals several molecular weightspecies including 98 kDa, 72 kDa 50 kDa and 35-40 kDa. Upon reductionthe following species are seen 68 kDA, 43 kDa and 20-22 kDA. Thenon-reduced 98 kDa species is comtemplated to comprise a homodimer oftwo 50 kDa subunits each comprising amino acids #28-#454 as shown inTable III. The 72 kDa species is contemplated to comprise a heterodimerof a 50 kDa subunit (comprising amino acids #28-#454 of Table III asdescribed above) and a 20 kDa subunit comprising amino acids #317-#454as shown in Table III. The 35-40 kDa species is contemplated torepresent the mature species comprising a homodimer of two 20 kDasubunits each comprising amino acids #317-#454 as shown in Table III.

C. BMP-5 Activity

BMP-5 (containing 100 ug/ml soluble heparin) purified in a preliminaryexperiment over octyl-sepharose (HIC) [see description below] then overheparin sepharose in a manner similar to the butyl then heparin stepsdescribed above is mixed with 20 mg rat matrix and implanted for 10 daysaccording to the rat ectopic assay described above in Example III.Approximately 1-3 ug BMP-5 protein from the heparin sepharose stepresults in the formation of cartilage and bone.

The octyl-SEPHAROSE purification step is carried out by adding solid(NH₄)₂ SO₄ to BMP-5 conditioned media containing 100 ug/ml solubleheparin to a final concentration of 1M. This is loaded onto a column ofoctly-SEPHAROSE equilabrated in 1M (NH₄)₂ SO₄, 50 mM Tris pH 7.4. Thecolumn is washed with starting buffer then with 50 mM Tris pH 7.4 andeluted with 50 mM Tris, 6M urea, 0.2% octly glucoside pH 7.4.Purification over heparin SEPHAROSE is by step gradient, washed with 50mM Tris, 0.15M NaCl, 6M urea pH 7.4, eluted with 50 mM Tris, 2M NaCl, 6Murea pH 7.4. The material implanted is 2M NaCl.

The foregoing descriptions detail presently preferred embodiments of thepresent invention. Numerous modifications and variations in practicethereof are expected to occur to those skilled in the art uponconsideration of these descriptions. Those modifications and variationsare believed to be encompassed within the claims appended hereto.

What is claimed is:
 1. A composition comprising an isolated and purifiedprotein having the amino acid sequence from amino acid #317 (Ala) toamino acid #454 (His) as shown in Table III said protein furthercharacterized by the ability to induce the formation of cartilage andbone.
 2. A composition comprising an isolated and purified proteinhaving the amino acid sequence from amino acid #323 (Asn) to #454 (His)as shown in Table III said protein further characterized by the abilityto induce the formation of cartilage and bone.
 3. A compositioncomprising an isolated and purified protein produced by the steps of(a)culturing a cell transformed with a vector comprising the DNA sequenceof Table III from nucleotide #1665 to #2060; and (b) recovering from theresulting culture medium said protein having the amino acid sequencefrom amino acid #323 (Asn) to amino acid #454 (His) as shown in TableIII said protein further characterized by the ability to induce theformation of cartilage and bone.
 4. A composition comprising a purifiedprotein produced by the steps of(a) culturing a cell transformed with avector comprising the DNA sequence of Table III from nucleotide #699 to#2060; and (b) recovering from the resulting culture medium a proteinhaving the amino acid sequence from amino acid #317 (Ala) to amino acid#454 (His) as shown in Table III said protein further characterized bythe ability to induce the formation of cartilage and bone.
 5. Thecomposition of claim 1 further comprising a matrix supporting saidprotein and providing a surface for bone and cartilage growth.
 6. Thecomposition of claim 5 wherein said matrix comprises a material selectedfrom the group consisting of hydroxyapatite, collagen, polylactic acidand tricalcium phosphate.
 7. A method for inducing bone and cartilageformation in a patient in need of same comprising administering to saidpatient an effective amount of the composition of claim
 1. 8. Acomposition comprising an isolated and purified protein having an aminoacid sequence selected from the group consisting ofa) amino acid #317(Ala) to #454 (His) of Table III; b) amino acid #323 (Asn) to #454 (His)of Table III; c) amino acid #1 (Met) to #454 (His) of Table III; and d)the amino acid sequence of Table I;said protein characterized by theability to induce the formation of cartilage and bone.
 9. A compositioncomprising an isolated and purified protein having the amino acidsequence from amino acid #1 (Met) to #454 (His) of Table III saidprotein characterized by the ability to induce the formation ofcartilage and bone.
 10. A composition comprising an isolated andpurified protein dimer with at least one of the subunits having theamino acid sequence from amino acid #317 (Ala) to amino acid #454 (His)of Table III said protein characterized by the ability to induce theformation of cartilage and bone.
 11. A composition comprising anisolated and purified protein dimer with at least one of the subunitshaving the amino acid sequence from amino acid #323 (Asn) to amino acid#454 (His) of Table III said protein characterized by the ability toinduce the formation of cartilage and bone.
 12. A composition comprisingan isolated and purified protein dimer with at least one of the subunitshaving the amino acid sequence from amino acid #28 (Gly) to amino acid#454 (His) of Table III said protein characterized by the ability toinduce the formation of cartilage and bone.
 13. A composition comprisingan isolated and purified protein produced by the steps of(a) culturing acell transformed with a vector comprising a DNA sequence having thesequence of Table III from nucleotide #1647 to #2060; and (b) recoveringfrom the resulting culture medium said protein the amino acid sequencefrom amino acid #317 (Ala) to amino acid #454 (His) as shown in TableIIIsaid protein characterized by the ability to induce the formation ofcartilage and bone.
 14. The composition of claim 3 wherein saidtransformed cell is mammalian.
 15. A composition comprising an isolatedand purified protein encoded by the DNA sequence encoding the BMP-5 ofATCC 68019 said protein characterized by the ability to induce theformation of cartilage and bone.
 16. A composition comprising anisolated and purified protein having the amino acid sequence of Table Isaid protein characterized by the ability to induce the formation ofcartilage and bone.
 17. The composition of claim 2 further comprising amatrix supporting said protein and providing a surface for bone andcartilage growth.
 18. The composition of claim 4 wherein saidtransformed cell is mammalian.
 19. The composition of claim 13 whereinsaid transformed cell is mammalian.
 20. A bone morphogenetic protein-5(BMP-5) produced by culturing a cell transformed with a vectorcomprising the DNA sequence of Table III from nucleotide #699 tonucleotide #2070 and recovering said protein from the resulting culturemedium.